Page | 1 Frequently Asked Questions GeneChip ® miRNA Arrays Affymetrix ® miRNA Array Plates Affymetrix ® miRNA Array Strips 1. Why can’t I see the miRBase identifier for probe sets in Affymetrix ® Expression Console™ Software charts and graphs? Affymetrix has changed the naming convention for probe sets on our miRNA arrays to be consistent with other array designs. Probe set IDs on Affymetrix’ miRNA 4.0 and 4.1 Arrays (and newer) will be a unique number. The probe set name will have the accession number and an Affymetrix suffix (e.g., “_st”). While the transcript ID is no longer contained in the probe set name, it is included in the annotation file. 2. Why are there more data headers and columns in my miRNA 4.0/4.1 or newer annotation files? The miRNA 4.0/4.1 annotations contain additional data fields to help ease the integration and correlation of small non-coding RNA data contained on Affymetrix’ miRNA 4.0 and 4.1 Arrays (and newer) with the content from our other expression products such as GeneChip® Human Genome U133 Plus 2.0 Array, GeneChip ® Human Gene 2.0 ST Array, and GeneChip ® Human Transcriptome Array 2.0. These additional fields include: Genomic context (where the miRNA is located in the genome) Clustered miRNA (miRNA that are located 10 KB from one another) Predicted miRNA targets Validated miRNA targets 3. Which assay do you recommend for use with Affymetrix’ GeneChip ® miRNA Arrays? Affymetrix only supports use of the Affymetrix ® FlashTag™ Biotin HSR RNA Labeling Kit for preparing targets to be applied to GeneChip ® miRNA Array, Affymetrix ® miRNA Array Strip, and Affymetrix ® miRNA Array Plate. This product may be ordered through Affymetrix using part numbers 901910 for the 10- reaction kit and 901911 for the 30-reaction kit. 4. Do we have to use any Affymetrix ® Reagents with our assay? Yes, the Affymetrix ® Reagents needed to process Affymetrix’ miRNA Arrays are listed below: P/N Description Qty_______ 900454 GeneChip ® Hybridization Control Kit 30 reactions 901910 Affymetrix ® FlashTag™ Biotin HSR RNA Labeling Kit 10 reactions 901911 Affymetrix ® FlashTag™ Biotin HSR RNA Labeling Kit 30 reactions In addition to the reagents listed above, you will need the appropriate hybridization, wash, and stain kit for your instrument. The three kits are listed below: P/N Description Qty_______ 902276 GeneTitan ® Hybridization, Wash, and Stain Kit for miRNA Array Plates 96 reactions 900720 GeneChip ® Hybridization, Wash, and Stain Kit 30 reactions 902134 GeneAtlas ® Hybridization, Wash, and Stain Kit for miRNA Array Strips 60 reactions
1. Why can’t I see the miRBase identifier for probe sets in Affymetrix® Expression Console™ Software charts and graphs?
Affymetrix has changed the naming convention for probe sets on our miRNA arrays to be consistent with other array designs. Probe set IDs on Affymetrix’ miRNA 4.0 and 4.1 Arrays (and newer) will be a unique number. The probe set name will have the accession number and an Affymetrix suffix (e.g., “_st”). While the transcript ID is no longer contained in the probe set name, it is included in the annotation file.
2. Why are there more data headers and columns in my miRNA 4.0/4.1 or newer annotation files?
The miRNA 4.0/4.1 annotations contain additional data fields to help ease the integration and correlation of small non-coding RNA data contained on Affymetrix’ miRNA 4.0 and 4.1 Arrays (and newer) with the content from our other expression products such as GeneChip® Human Genome U133 Plus 2.0 Array, GeneChip® Human Gene 2.0 ST Array, and GeneChip® Human Transcriptome Array 2.0. These additional fields include:
Genomic context (where the miRNA is located in the genome) Clustered miRNA (miRNA that are located 10 KB from one another) Predicted miRNA targets Validated miRNA targets
3. Which assay do you recommend for use with Affymetrix’ GeneChip® miRNA Arrays?
Affymetrix only supports use of the Affymetrix® FlashTag™ Biotin HSR RNA Labeling Kit for preparing targets to be applied to GeneChip® miRNA Array, Affymetrix® miRNA Array Strip, and Affymetrix® miRNA Array Plate. This product may be ordered through Affymetrix using part numbers 901910 for the 10-reaction kit and 901911 for the 30-reaction kit.
4. Do we have to use any Affymetrix® Reagents with our assay?
Yes, the Affymetrix® Reagents needed to process Affymetrix’ miRNA Arrays are listed below:
P/N Description Qty_______
900454 GeneChip® Hybridization Control Kit 30 reactions
In addition to the reagents listed above, you will need the appropriate hybridization, wash, and stain kit for your instrument. The three kits are listed below:
P/N Description Qty_______
902276 GeneTitan® Hybridization, Wash, and Stain Kit for miRNA Array Plates 96 reactions
900720 GeneChip® Hybridization, Wash, and Stain Kit 30 reactions
902134 GeneAtlas® Hybridization, Wash, and Stain Kit for miRNA Array Strips 60 reactions
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5. Why do Affymetrix® miRNA Array Strips and miRNA Array Plates have different hybridization, wash, and stain kits from Affymetrix® 3’IVT Array Strips and 3’ IVT Array Plates and Affymetrix® Gene ST Array Strips and Gene ST Array Plates?
IMPORTANT: The hybridization, wash, and stain protocol for Affymetrix® miRNA Array Strips and Array Plates is different from the 3’ IVT and the Gene ST Array Strips and Array Plates protocols. Additional reagents have been added to these kits to help ensure adequate washing after the stain has been introduced to the array. A new quick reference card, which has been created to aid you in filling the wash/stain tray, can be found at the URL below: http://www.affymetrix.com/Auth/support/downloads/quick_reference_cards/geneatlas_tray_filling_mirna_qrc
6. What instruments and software will be supported by Affymetrix?
The following instruments and software will be supported by Affymetrix for processing GeneChip® miRNA Arrays:
o GeneChip® Scanner 3000 7G and 7G Plus o GeneChip® Fluidics Station 450 o GeneChip® Hybridization Oven 640 and 645 o GeneChip® Command Console® Software (AGCC)
The following instruments and software will be supported by Affymetrix for processing Affymetrix® miRNA Array Strips:
o GeneAtlas® System o GeneAtlas® Instrument Control Software (version 1.0.6 or higher)
The following instruments and software will be supported by Affymetrix for processing Affymetrix® miRNA Array Plates:
o GeneTitan® Instrument
The following analysis software packages are supported by Affymetrix for processing Affymetrix' miRNA Arrays, miRNA Array Strips, and miRNA Array Plates:
o Expression Console™ Software o Transcriptome Analysis Console™ Software
7. Can I analyze my Affymetrix miRNA Array Strip data using Partek® Express™?
Yes, Partek Express version 1.12.1121 supports the analysis of miRNA Array Strips.
8. Can I use the human, mouse, and rat .ps files to limit my data output in Partek® Express™ and Partek® Genomics Suite?
No, Partek Express and Partek Genomics Suite do not use the .ps file when analyzing data. We will notify you when this has been resolved.
9. Why do I need to use the Control Oligo B2 with my assay?
Control Oligo B2 (3 nM) is a pre-labeled DNA spike-in control required for hybridization to the control probes on the array utilized for grid alignment by AGCC. A failure to include Control Oligo B2 in the hybridization cocktail will result in the inability of the software to apply a grid over the scanned image, leading to the unrecoverable loss of sample data from the array. The absence of the Control Oligo B2 also voids the customer's ability to submit an array replacement request to Affymetrix.
10. Why do I need to use GeneChip® 20X Hybridization Controls?
The Hybridization Controls are high-quality controls for monitoring array hybridization, washing, and staining for reproducible results.
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20X Hybridization Controls are composed of a mixture of biotinylated and fragmented cRNA of bioB, bioC, and bioD from E. coli and cre from P1 bacteriophage in staggered concentrations. The premixed controls are ready to be added directly to the hybridization cocktail. Probes for detecting these controls are present on GeneChip® miRNA Array, Affymetrix® miRNA Array Strip, and Affymetrix® miRNA Array Plate.
The 20X Hybridization Controls are spiked into the hybridization cocktail, independent of RNA sample preparation, and are thus used to evaluate sample hybridization efficiency on eukaryotic gene expression arrays.As the 20X Hybridization Controls are used by Affymetrix to troubleshoot potential array and array processing issues, a failure to include the 20X Hybridization Controls in the hybridization cocktail will void the customer's ability to submit an array replacement request to Affymetrix.
11. Where can I find the library files for Affymetrix’ miRNA Arrays ?
There are two ways to access the library files for the miRNA arrays:
Access the library files on the support page: http://www.affymetrix.com/support/technical/libraryfilesmain.affx
The library files can be downloaded from the Affymetrix’ Array product web pages at www.affymetrix.com.
For GeneAtlas System you can also download the library files using GeneAtlas® Instrument Control Software by going to the Utility Actions menu, selecting Download Library Files, and selecting the appropriate library file package before proceeding to sample registration.
12. What data analysis software solutions are offered by Affymetrix?
Affymetrix provides both Expression Console™ Software for normalization and signal summarization of your experimental data and Affymetrix® Transcriptome Analysis Console Software for differential expression analysis and visualization free of charge. Due to the presence of multiple organisms on the miRNA arrays, the chromosomal location feature in Transcriptome Analysis Console Software will not give accurate results.
For analysis in Transcriptome Analysis Console Software, array type-specific configuration and annotation files are required. Use the “Download Array Type Files” button in the Preferences tab to download and install array type-specific library files.
13. How do I use the new .ps files for limiting the data output to human, mouse, or rat probe sets in Expression Console Software?
To use the new .ps files, you must download the Affymetrix® miRNA library analysis file package from the product or support web page and place all files in the folder used for Expression Console analysis library files. Restart Expression Console Software (if open) and reload the existing study, or create a new study.
When you click the “Run Analysis” button, a pop-up window will appear and provide you with a listing of available analyses. These include 1) All probe sets for all organisms, 2) Human only probe sets, 3) Mouse only probe sets, 4) Rat only probe sets. All four of these analysis options include the choice of RMA+DABG with and without normalization. Figure 1 is an example of what should be expected if the new analysis files were added properly.
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14. Which fluidics script is required with GeneChip® miRNA Array?
The following fluidics scripts should be used with each version of GeneChip miRNA Array:
• GeneChip® miRNA Array FS450_0003
• GeneChip® miRNA 2.0 Array FS450_0003
• GeneChip® miRNA 3.0 Array FS450_0002
• GeneChip® miRNA 4.0 Array FS450_0002
15. What is the recommended temperature for hybridization?
The following hybridization temperatures should be used with each version of Affymetrix’ miRNA Array:
• GeneChip® miRNA Arrays 48º C
• Affymetrix® miRNA Array Plates 48º C
• Affymetrix® miRNA Array Strips 48º C
16. What is the shelf life for Affymetrix’ miRNA Arrays?
This product has 12 months of shelf life with a minimum shelf life of 3 months. Minimum shelf life is defined as the minimum amount of time from date of order shipment by Affymetrix to array expiration date. If users receive products with shorter than expected minimum shelf life, Affymetrix will replace the products at no cost to the user—subject to terms and conditions.
17. What are the proper storage conditions for Affymetrix’ miRNA Arrays?
18. Where can I get a list of miRNAs represented on Affymetrix’ miRNA Arrays?
You can get the list by downloading the annotation files that will be available on the www.affymetrix.com product web page.
19. What is the minimum amount of total RNA for this assay?
Minimum is 130 ng; maximum is 1,000 ng total RNA.
For more information about the assay, refer to the following link: http://media.affymetrix.com/support/help/faqs/flashtag/flashtag_faqs.pdf
20. What is the hybridization volume recommended for Affymetrix’ miRNA Arrays?
GeneChip® miRNA Array 130 μL of hybridization cocktail
Affymetrix® miRNA Array Plates 120 μL of hybridization cocktail
Affymetrix® miRNA Array Strips 120 μL of hybridization cocktail
21. What is the recommended hybridization time for Affymetrix’ miRNA Arrays?
GeneChip® miRNA Array 17 +/- 1 hour
Affymetrix® miRNA Array Plates 17 +/- 1 hour
Affymetrix® miRNA Array Strips 20 +/- 1 hour
22. What is the orientation of the probes on the array?
Probes on the array detect sense target.
23. What is the difference between GeneChip miRNA Array, Affymetrix miRNA Array Strip, and Affymetrix miRNA Array Plate?
The only difference is the form factor of the array. The arrays have the exact same design and contain the same number of probes and probe sets.
24. Can I combine the CEL files from GeneChip miRNA Arrays, Affymetrix miRNA Array Strips, and Affymetrix miRNA Array Plates into the same experiment?
No, the CEL files from GeneChip miRNA Array, Affymetrix miRNA Array Strip, and Affymetrix miRNA Array Plates are not compatible.
25. What is the tolerance around hybridization time?
Affymetrix recommends maintaining consistency in array processing protocols to minimize introduction of variability. Our recommended hybridization time for Affymetrix® miRNA Array Strip is 20 hours. Affymetrix miRNA Array Strip should be hybridized no less than 20 hours and no longer than 24 hours. We strongly encourage maintaining consistent hybridization times to avoid false positives with respect to differential expression due to hybridization time.
26. How long can I store my array strip after hybridization before starting the wash/stain protocol?
We recommend storing the hybridized array at 4° C in Wash A Buffer for no longer than 4 hours prior to starting the wash/stain protocol. Equilibrate array strip to room temperature before proceeding.
27. How long can I store my array strip after the wash/stain protocol before scanning?
The arrays may be stored at 4° C in Array Holding Buffer for up to 24 hours prior to scanning with negligible impact on data. Equilibrate array strip to room temperature before scanning.
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28. How do results from GeneChip® miRNA Arrays compare to Affymetrix® miRNA Array Plates?
When evaluating non-control probe sets common to both arrays, we observed very high signal and fold change correlation for GeneChip® miRNA 3.0 Arrays compared to Affymetrix® miRNA 3.1 Array Plates (Pearson correlation coefficients of 0.97−0.98 for signal correlation and Pearson correlation coefficients of 0.96−0.98 for fold change correlation). Examples are shown in Figures 2, 3, 4, and 5.
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Figure 2: Median RMA signal correlation for detecting human miRNA probe sets in brain.
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Figure 3: Median fold change correlation for detecting human miRNA for brain compared to lung.
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Figure 4: Median RNA signal correlation for detecting human miRNA, precursor miRNA, and sno/scaRNA probe sets in brain.
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Figure 5: Median fold change correlation for detecting human miRNA, precursor miRNA, and sno/scaRNA probe sets for brain compared to lung.
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29. What kind of dynamic range can I expect from Affymetrix® miRNA Array Plates?
Affymetrix® miRNA 3.1 Array Plate provides a similar dynamic range to GeneChip® miRNA Array. Figure 6 shows the linear dynamic range of Affymetrix miRNA 3.1 Array Plate over 4 log10. The data in Figure 6 represents 47 different miRNAs spiked into 130 ng of human liver total RNA.
30. How are sno/sca and hairpin probe sets selected and how does that impact my signal summarization?
Probes for snoRNA, scaRNA, and hairpins are selected to maximize probe response to target concentration in the sample while minimizing cross-hybridization to other potential targets in the sample. In order to minimize cross-hybridization, potential targets are inferred from the landscape of known sequences from the input dataset and used in a filtering process called pruning which penalizes probe candidates that may cross-hybridize to unwanted targets. As the landscape of known sequences improves, we can improve our pruning set to avoid probe candidates previously thought to be unique. An improved pruning set will reduce the chances a probe will cross-hybridize to unwanted target, improving the probe set representation of the intended target. As a result of the improved probe selection, few probe sets are required to represent the same number of sequences.
31. What signal and fold change concordance can I expect between Affymetrix’ miRNA 4.0 and miRNA 4.1 Arrays and miRNA 3.0 and miRNA 3.1 Arrays?
You should expect high signal and fold change concordance for transcripts with identical probes on the miRNA 4.0 and miRNA 4.1 Arrays and the miRNA 3.0 and miRNA 3.1 Arrays. As noted in question #30, the snoRNA, scaRNA, and precursor miRNA probe sets were reselected, and for some probe sets in these categories, the probes are not identical between the two array designs. Because the probes selected may be different, one may observe changes in the probe set signal summary for a given target. This is
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Figure 6: Dynamic range.
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possible as different probe sequences will have varying affinity to the target and may show a higher or lower absolute probe signal depending on the GC content of the probes. However, fold changes are typically well-correlated to the legacy probe set unless there is cross-hybridization in the legacy probe set. Our expectation is that more unique and specific probes result in more specific hybridization to the intended target, and therefore a better performing probe set.
Figures 7–10: Signal and fold change correlation between GeneChip® miRNA 3.0 Array and GeneChip® miRNA 4.0 Array matching probe sets and matching probe sets with identical probe sequences (human only).
Target preparation replicates of human brain and lung samples were pooled and hybridized to four miRNA 3.0 Arrays and four miRNA 4.0 Arrays. The Pearson product moment correlation was calculated from the median signal of matched probe sets and from the median fold change of detected matched probe sets on the miRNA 3.0 Array compared to the miRNA 4.0 Array. Probe sets were defined as detected if the median DABG p-value of the four miRNA 3.0 Array brain replicates was less than 0.06.
Two subsets of matched probe sets were used for miRNA 3.0 Array to miRNA 4.0 Array signal and fold change correlation: All human probe sets including those that do not necessarily share identical probe sequences and human probe sets for which the probe sequences are identical on both the miRNA 3.0 and miRNA 4.0 Arrays.
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Figure 7: Signal correlation (Human brain) from 5,070 matching probe sets. (5.8s rRNA: red points, Affymetrix control sequences: dark blue points, CDBox RNA: yellow points, HAcaBox RNA: black points, spike-in controls: light blue points, miRNA: green points, scaRNA: grey points, precursor miRNA: pale blue points).
Figure 8: Signal correlation (Human brain) from 1,886 matching probe sets with identical probe sequences. (5.8s rRNA: red points, Affymetrix control sequences: dark blue points, CDBox RNA: yellow points, HAcaBox RNA: black points, spike-in controls: light blue points, miRNA: green points, scaRNA: grey points, precursor miRNA: pale blue points).
Figure 9: Signal correlation (Human lung) from 5,070 matching probe sets. (5.8s rRNA: red points, Affymetrix control sequences: dark blue points, CDBox RNA: yellow points, HAcaBox RNA: black points, spike-in controls: light blue points, miRNA: green points, scaRNA: grey points, precursor miRNA: pale blue points).
Figure 10: Signal correlation (Human lung) from 1,886 matching probe sets with identical probe sequences. (5.8s rRNA: red points, Affymetrix control sequences: dark blue points, CDBox RNA: yellow points, HAcaBox RNA: black points, spike-in controls: light blue points, miRNA: green points, scaRNA: grey points, precursor miRNA: pale blue points).
Figure 11: Fold change correlation (Human lung) from 5,070 matching probe sets. (5.8s rRNA: red points, Affymetrix control sequences: dark blue points, CDBox RNA: yellow points, HAcaBox RNA: black points, spike-in controls: light blue points, miRNA: green points, scaRNA: grey points, precursor miRNA: pale blue points).
Figure 12: Fold change correlation (Human lung) from 1,886 matching probe sets with identical probe sequences. (5.8s rRNA: red points, Affymetrix control sequences: dark blue points, CDBox RNA: yellow points, HAcaBox RNA: black points, spike-in controls: light blue points, miRNA: green points, scaRNA: grey points, precursor miRNA: pale blue points).
